Induction, Purification, CDNA Gene Cloning And Expression Of Laccase In Pholiota Namek

Laccases are multi-copper proteins that use molecular oxygen to oxidize various aromatic andnon-aromatic compounds. Their widespread distribution in fungi has been verified for many years. Its abilityto degrade lignin and many toxic substances such as ployphenolic materials brings laecases a wideapplication in pulp and paper production, textile dye bleaching and environment protection. In thisdissertation, the culture and induction conditions, purification and characterization, cDNA gene cloning andheterogenous expression of pholiota namek laccase were studied.Comparing four culture media A, B, C, D, which are usually used in laccase induction, D was the bestone and was used as basic culture media in further study. The proportion of carbon and nitrogen sources waschanged to study its effects on laccase production. HNHC (high nitrogen source and high carbon source)culture medium can lead to both the highest laccase activity and the best mycelial growth. Asparagine was thebest nitrogen sources compared with ammonium nitrate, carbamide and yeast extract.Extracellular laccase formation could be greatly influenced by the addition of Cu²⁺and Mn²⁺.Supplementing the growth medium with low concentration Cu²⁺(no more than lmmol/L) leads tosignificantly enhanced laccase activity. A decrease in laccase activity level was obtained in the presence ofhigh concentration Cu²⁺(above 1mmol/L). Mn²⁺ had little effect on laccase activity when added at lowconcentration, but high concentration (above 1mmol/L) addition of/Mn²⁺ could obviously inhibit laccaseproduction.DEAE-Sepharose^TMCL-6B,Sephacryl^TMS-100 are used to purify lacease from the liquid medium andthe effects of purification was evaluated by SDS-PAGE. The optimum pH and temperature of partiallypurified laeease for oxidizing guaiacol are 5.5 and 55℃, respectively. The enzyme shows a good stabilityfrom 40℃to 60℃. The stability of lacease decreased severely at a temperature above 60℃.Degenerated primers are designed based on the four conserved Cu²⁺ binding sites and a fragment oflaccase cDNA was obtained by RT-PCR. SMART^TM RACE cDNA Amplification Kit was used to get thefull-length cDNA of laccase. The full length of the cDNA was 1733bp, containing an open reading frame of1524bp encoding for 514 amino acid, among which the former 18 amino acids belongs to the signal peptide.The theoretical pâ… and the molecular mass are 5.52 and 56952.90Da respectively.EasySelect^TM Piehia Expression Kit of invitrogen was used for the heterogenous expression. PPiczαB is used as expression vector and KM71H(Mut^s, Arg⁺) as host strain. The positive clones we got from electronictransformation are tested in both MM plates(with Cu²⁺and ABTS) and shaken-flask cultivation(cultured inBMMY), methanol was added every day. Although DNA sequencing and many other molecular biologicalmethods have verified the existence of the laccase gene in yeast genome, the expression conditions and otherfactors should be studied to complete the heterogenous expression.